Immunomodulation of brassinosteroid functions in seeds of Arabidopsis thaliana (L.) [Elektronische Ressource] / von Tatiana Ankudo
80 Pages
English
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Immunomodulation of brassinosteroid functions in seeds of Arabidopsis thaliana (L.) [Elektronische Ressource] / von Tatiana Ankudo

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80 Pages
English

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Immunomodulation of Brassinosteroid Functions in Seeds of Arabidopsis thaliana (L.) Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr.rer.nat.) vorgelegt der Mathematisch-Naturwissenschaftlich-Technischen Fakultät (mathematisch-naturwissenschaftlicher Bereich) der Martin-Luther-Universität Halle-Wittenberg von Frau Tatiana Ankudo geb. am: 11.07.1976 in: Minsk, Weissrussland Gutachter: 1. Prof. Dr. habil. Werner Roos, Halle 2. Dr. Udo Conrad, IPK Gatersleben 3. Dr. Thomas Altmann, MPI Golm Halle (Saale), 21.01.2004 urn:nbn:de:gbv:3-000006137[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000006137]LIST OF ABBREVIATIONS List of abbreviations ALP .........................alkaline phosphatase ABA ........................abscisic acid ramp .........................ampicillin resistance anti-Bl scFv..............anti-brassinolide single-chain Fv antibodies BA ...........................6-Benzyladenine BAK1.......................BRI1 associated receptor kinase1 Bl.............................brassinolide BRI1 ........................brassinosteroid-insensitive BRs..........................brassinosteroids BSA .........................bovine serum albumin c-myc.......................c-myc tag sequence CaMV35S ................cauliflower mosaic virus 35S promoter Cs.............................castasterone 24-Cs .......................24-epicastasterone E.coli.

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Published 01 January 2004
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Dissertation 
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr.rer.nat.)  vorgelegt der
Mathematisch-Naturwissenschaftlich-Technischen Fakultät (mathematisch-naturwissenschaftlicher Bereich) der Martin-Luther-Universität Halle-Wittenberg
 Immunomodulation of Brassinosteroid Functions in Seeds of Arabidopsis thaliana (L.)                               Gutachter:  1.Prof. Dr. habil. Werner Roos, Halle   2.Dr. Udo Conrad, IPK Gatersleben   3.Dr. Thomas Altmann, MPI Golm    Halle (Saale),21.01.2004   
von Frau Tatiana Ankudo geb. am: 11.07.1976 in: Minsk, Weissrussland
urn:nbn:de:gbv:3-000006137 [http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000006137]
List of abbreviations
LIST OF ABBREVIATIONS
    ALP ......................... alkaline phosphatase ABA ........................ abscisic acid r amp ......................... ampicillin resistance anti-Bl scFv.............. anti-brassinolide single-chain Fv antibodies BA ........................... 6-Benzyladenine BAK1....................... BRI1 associated receptor kinase1 Bl ............................. brassinolide BRI1 ........................ brassinosteroid-insensitive BRs .......................... brassinosteroids BSA ......................... bovine serum albumin c-myc ....................... c-myc tag sequence CaMV35S ................ cauliflower mosaic virus 35S promoter Cs............................. castasterone 24-Cs ....................... 24-epicastasterone E.coli........................Escherichia coli EBL ......................... 24-epibrassinolide ELISA...................... enzyme-linked immunosorbent assay ER............................ endoplasmic reticulum GA ........................... gibberellic acid GC-MS .................... gas chromatography-mass spectrometry GFP.......................... green fluorescent protein GST ......................... glutathione S-transferase HSPs ........................ heat-shock proteins JA ............................ (3R,7R)-jasmonic acid JAVII ....................... transgenic plants with anti-JA scFv targeted into the cytosol KDEL ...................... retention sequence LEA ......................... late embryogenic abundant LeB4 ........................ legumin B4 promoter LeB4SP.................... legumin B4 signal sequence MS ........................... Murashige-Skoog NAA ........................ 1-Naphtalene acetic acid ori ............................ origin of replication PB ............................ protein body
LIST OF ABBREVIATIONS
  PCR ......................... polymerase chain reaction RLKs ....................... receptor-like protein kinases poly A ...................... polyadenylation signal RP ............................ ribosomal proteins rpm .......................... revolutions per minute PSV.......................... protein storage vacuole scFv ......................... single-chain variable fragment sp ............................. signal peptide TAK16 .................... transgenic plants with anti-Bl A16 scFv expression in ER under control of LeB4 promoter TBU16 ..................... transgenic plants with anti-Bl A16 scFv expression in ER under control of USP promoter TSP .......................... total soluble protein USP.......................... unknown seed-specific promoter WT........................... wild type
 
CONTENTS 
Contents  1. Introduction  1.1. The history of brassinosteroids .....................................................................1 1.2. Natural occurrence of brassinosteroids in the plant kingdom ........................2 1.3. Biosynthesis.................................................................................................3 1.4. Physiological action of brassinosteroids .......................................................5 1.5. Molecular genetic of brassinosteroid action ..................................................7 1.5.1.    Brassinosteroid signal transduction..............................................................7 1.5.2. Brassinosteroid regulated gene expression................................................. 10 1.6. Immunomodulation of hormone functions in plant cells ............................ 10 1.7. Aim of the work ........................................................................................ 12  2. Materials and methods  2.1. Materials ................................................................................................... 13 2.1.1. Bacterial strains and phages ...................................................................... 13 2.1.2. Vectors...................................................................................................... 13 2.1.3. ScFv genes in pIT vector........................................................................... 13 2.1.4. Plant material ............................................................................................ 13 2.1.5. Oligonucleotide primers ............................................................................ 13 2.1.5.1. Oligonucleotide primers for PCR amplification ......................................... 13 2.1.5.2. Oligonucleotide primers for DNA sequencing ........................................... 14 2.1.6. Media........................................................................................................ 14 2.1.6.1. Media for plant culture .............................................................................. 14 2.1.6.2. Media for bacterial culture ........................................................................ 14 2.1.7. Buffers ...................................................................................................... 15 2.1.8. Enzymes ................................................................................................... 17 2.1.9. Antibiotics ................................................................................................ 17 2.1.10. Immunochemicals ..................................................................................... 18 2.1.11. DNA- and proteinmarkers ......................................................................... 18 2.1.12. Plant hormones ......................................................................................... 18
CONTENTS 
2.1.13. Kits ......................................................................................................... 18 2.1.14. Special laboratory reagents...................................................................... 18 2.1.15. Special laboratory tools ........................................................................... 19 2.1.16. Special laboratory equipment .................................................................. 19 2.1.17. Software.................................................................................................. 19  2.2. Methods  2.2.1.E.coli 20transformation ............................................................................... 2.2.1.1. Preparation of competentE.colicells....................................................... 20 2.2.1.2. Heat shock transformation of competentE.colicells................................ 20 2.2.1.3.   Electroporation........................................................................................20 2.2.2. Transformation ofAgrobacterium tumefaciens 20cells ................................ 2.2.2.1. Preparation of competentAgrobacterium tumefacienscells ..................... 20 2.2.2.2. Cold shock transformation of competentAgrobacterium tumefaciens  cells ........................................................................................................ 20 2.2.3. Production of soluble scFv fromE.coli.................................................... 21 2.2.4. Purification of scFv from plant extract..................................................... 21 2.2.5. ELISA with soluble anti-epibrassinolide scFv antibodies in bacterial supernatant and purified from plant extract ............................................. 21 2.2.6. Western blot analysis .............................................................................. 22 2.2.7. Affymetrix analysis ................................................................................. 22 2.2.7.1. RNA preparation ..................................................................................... 22 2.2.7.2. Affymetrix hybridization......................................................................... 22 2.2.7.3. Expression analysis ................................................................................. 22 2.2.8. Seed germination experiment .................................................................. 22 2.2.9. Plasmid preparation................................................................................. 22 2.2.10. Extraction of plant genomic DNA ........................................................... 23 2.2.10.1. The rapid plant DNA extraction, PCR grade............................................ 23 2.2.10.2. Extraction of high purity plant DNA ....................................................... 23 2.2.11. Gene expression analysis......................................................................... 23 2.2.12. Construction of expression cassettes for plant transformation .................. 23 2.2.13. Colony PCR of transgenicAgrobacterium tumefacienscells ................... 25 2.2.14. Transformation ofNicotiana tabacum..................................................... 25 
CONTENTS 
2.2.14.1. Leaf disk method.....................................................................................25 2.2.14.2. Propagation of transgenic plants .............................................................. 25 2.2.15. Plant transformation ofArabidopsis thalianaby floral dip....................... 25 2.2.16. Characterization of transgenic plants ....................................................... 26 2.2.16.1. Characterization of transgenic plants by western blot analysis ................. 26 2.2.16.2. Quantitative detection of scFv expression by western blot analysis.......... 26 2.2.17. Electron microscopy................................................................................ 26  3. Results  3.1. Construction of scFv-expression cassetes for plant transformation........... 27 3.2. Production of transgenic plants................................................................ 31 3.3. Western blot analysis of scFv expression in primary transgenic plants..... 32 3.4. Isolation of heterozygous lines ................................................................ 34 3.5. ELISA of protein extract from the seeds .................................................. 36 3.6. Antibody expression during the seed development .................................. 37 3.7. Ultrastructurial changes in transgenicAa rsiibsopdseeds ......................... 38 3.8. Developmental changes caused by the expression of anti-Bl scFv  antibodies................................................................................................ 40 3.8.1 Growth ...........................................................................................in vitro40 3.8.2. Growthin vivo........................................................................................ 43 3.9. Gene expression analysis during seed germination using Affymetrix GeneChip................................................................................................ 43 3.10. Genes with altered expression in the transgenic lines............................... 49 3.11. Seed protein content during germination ................................................. 50  4. Discussion............................................................................................... 52  5. Abstract................................................................................................... 58  6. Zusammenfassung................................................................................... 60  7. References .............................................................................................. 62  
CONTENTS 
8. Acknowledgements .................................................................................
 
 Declaration
  Curriculum vitae
71
 
INTRODUCTION  
1. Introduction  1.1. The history of brassinosteroids  Brassinosteroids (BRs) have been generally accepted as steroidal plant hormones only recently. Since the discovery of brassinolide (Bl) (Figure 1), isolated from rape (Brassica napus L.) in 1979 by Grove et al. extensive studies have been undertaken worldwide on this notable substance. Diverse species of plants have been found to contain Bl. Their characteristic physiological effect on growth and development of plants as well as their potential abilities in agricultural applications have started to be examined. These compounds were grouped in a new class of plant hormones. Before the isolation of Bl, long discussion had been carried out, whether steroidal hormones exist in plants or not. In the year of 1979, when the isolation of Bl was reported, such doubts came to the end, because Bl has a steroidal structure and has apparent hormonal properties. After the Groves report, castesterone (Cs) was isolated as the second BRs at the University of Tokyo (Yokota et al., 1982). Since then, a number of related steroidal compounds have been isolated from a variety of plant sources. To date, more then 40 free BRs and 4 BR conjugates have been found and fully characterized by spectrometric methods. Presumably, there is a number of yet unknown BRs and BR conjugates in plants. BRs have been found in at least 44 plant species. Some data suggest that BRs are more widely distributed than presently established. These findings suggest, that BRs are ubiquitously distributed in the plant kingdom. At first, investigations were focused on describing the biosynthetical pathways of BRs and their counterparts in plants. Recently, the major part of the biosynthesis of Bl has been established (Fujioka and Sakurai, 1997). This hypothetical model is based on information from extensive step-wise feeding studies performed with radiolabelled precursors of Bl and analysis of their bioconversion products. In the last decade, attention concentrated also to the physiological properties of BRs (Sakurai and Fujioka, 1993; Adam et al., 1996, Fujioka and Sakurai, 1997; Sasse, 1997; Yokota, 1997). High interest in the function of BRs has been elicited through the strong responses of intact plants and explants observed upon application of exogenous BRs. They were considered as promising compounds for application in agriculture, because they showed different regulatory activities on growth and development in plants. Because BRs affect a broad range of biological activities, it has long been difficult to determine their functions in plant growth with a simple treatment. Although the BRs are present in plant tissues, their importance for the regulation of plant growth and development has not been accepted as wide as for “classical” plant hormones such as auxins, gibberellins, cytokinins, jasmonates, ethylene and abscisic acid (Kende and Zeevaart, 1997). This situation changed dramatically with several reports on the identification and characterization of BR biosynthesis and signal transduction mutants (Clouse, 1996, 1997; Yokota, 1997). The phenotypes of the BR-deficient mutants presumed, that BRs are essential hormones and take part in light-regulated development. A new attempt to identify BR-insensitive mutants ofAarspsiibod resulted in the isolation of the dwarf mutant namedbri1 et al., 1996; Szekeres et al., (Clouse 1996). BRI1 was found to have strong homology to leucine-rich receptor kinases. Recent analysis of a range of BR biosynthesis and insensitivity mutants in Arabidopsisdecisive in discovering the physiological importance of BRs. was Although we have a limited knowledge about mode of action of BRs, research is  - 1 -   
 INTRODUCTION
moving rapidly along three convergent lines of analysis. Microchemical techniques are revealing the details of biosynthesis, distribution, and metabolism. The analysis of BR-deficient and BR-insensitive mutants aims to characterize the physiological roles of BRs in growth and development. The cloning of BR-regulated genes is providing insight into the molecular mechanisms of plant steroid hormone action. In future, the research of BRs will become increasingly important in understanding the plant growth regulation.  
 
 Figure 1.Brassinosteroids structure.   1.2. Natural occurrence of brassinosteroids in the plant kingdom Among plant steroids only BRs are ubiquitously distributed throughout the plant kingdom. They play essential roles in modulating the growth and differentiation of cells at nanomolar to micromolar concentrations (Clouse and Sasse, 1998). All BRs are hydroxylated derivatives of cholestane and, given the possibilities of combinations of substructures in rings A and B and the side chain, the family has many more members then it is reported to date. The compounds can be classified as C27, C28, or C29BRs, depending on the pattern of the side chain (Figure 1). BRs have been found in a wide range of plant species, in algae and pteridophytes, and three families of gymnosperms; in angiosperms, they have been shown to occur in 16 families of dicots, and 15 of monocots (Clouse and Sasse, 1998). They were detected in various plant parts such as pollen, seeds, leaves, roots, stems and flowers. Thus, BRs are widely distributed in the plant kingdom, and they are possibly biosynthesized in all parts of plant organs. Certainly they occur in shoots and seeds of the important experimental plantArabidopsis thaliana et al., 1996, (Fujioka Schmidt et al., 1997). Levels of endogenous BRs vary among plant tissues. The highest measured concentration is about 10-1nmol g-1fresh weight (Bl in the pollen ofBrassica napusandVicia faba) and the lowest is about 10-7 g nmol-1 (homocastasteron in immature seeds and sheaths of Chinese cabbage,rassBci a campestris var. pekinensis, et al., 2000). Pollen and immature seeds, the Khripach original so1gx ,1-0g  no  f,010a omnustve lowerually hanomrw se htignar oes1f f ,iwels ohots an leaves uss ceur al, Bofnoaterc  ghtninist oe moe hof th-1-100ng x g- w fw (Takatsuto, 1994). The average steroid content in mature seeds of sispodibarA  data of et al. (1998): brassinolide, 3,9x10-3 t nhmaloila nga- 1105xtsatsac ,9 ,noretrated by re;centsii llsu-4jugF l mio-1oler3x, y;p tstha01ako-3 nmol g-1; 6- n  - 2 -
 INTRODUCTION
deoxocastasterone, 3,5x10-3 nmol g-1; 6-deoxotyphasterol, 2,1x10-3 g nmol-1; 6-deoxoteasterone, 1,2x10-3 nmol g-1. Bioassay results suggest that roots also contain BRs, however, possibly due to their low concentration in the tissue, they were not isolated yet (Clouse and Sasse, 1998).  1.3. Biosynthesis Plant sterols have been extensively studied in the past years with a major focus on biosynthetic and biochemical aspects. Elucidation of BR biosynthesis and metabolism is fundamental to understand how plants regulate the endogenous level of active BR for their proper growth and development. Until recently, pathways of BR biosynthesis have remained unclear. Now Bl, the most important BR in plants, has been shown to be synthesized via two pathways from campesterol (Figure 2). Sterols are synthesized via the mevalonate pathway of isoprenoid metabolism. BRs must be converted from certain components of plant sterols, similar to animal and insect steroidal hormones which are derived from cholesterol. Among plant sterols, campesterol and its analogues are assumed to be the biosynthetic precursors of Bl, s hesis were ibnavseesdt iogna ttehde  uisdienngt ifteye doifn tgh oe f scidelel- cchualtiunr essk eolfe tCo.n .r oEsaerulsynt biof Blps os ety002 ,.la htiw )0uj(F t  ekaio2H-labeled 6-oxocampestanol, 6-deoxocastasterone and 6α-hydroxycastasterone, and the metabolites were analyzed by gas chromatography-mass spectrometry (GC-MS). The cell cultures produced representatives of C28 such as Bl, Cs, typhasterol, BRs, testasterone and cathasterone (Choi et al., 1996, Fujioka et al., 1995). The levels of BRs in cell cultures ofC. roseusbe comparable to those of BRs-richwere found to plant tissues such as pollen and immature seeds. Using the cell culture system ofC. roseus, biosynthetic studies of Bl were carried out by GC-MS analysis of the metabolites obtained from feeding labeled substrates. Campesterol as the major plant sterol was found in cell cultures. To investigate the metabolites of this steroid, labeled campesterol was effectively prepared from labeled mevalonic acid. By feeding the cells with radioactive campesterol, campestanol, 6α-hydroxycampesterol, and 6-oxocampestanol were identified as metabolites. Subsequent feeding experiments with labeled campestanol and 6αhydroxycampesterol revealed the biosynthetic sequence from campesterol to- 6-oxocampestanol as shown in Figure 2 (Suzuki et al., 1995). This sequence constitutes an early part of the biosynthetic pathway of Bl, since the introduction of vicinal hydroxyls in the side chain of 6-oxocampestanol will yield teasterone. Therefore, a biosynthetic pathway for BRs via hydroxylations and epimerisation after the oxidation at C6 of the B-ring was suggested. This pathway leading to Bl was proposed as an early C6-oxidation pathway. Transformation of 6-oxocampestanol to teasterone was described using chemically synthesized specimens of possible intermediates as probes. As a result, 22α-hydroxy-6-oxocampestanol was identified and named cathasterone (Fujioka et al., 1995) In the same work cathasterone was identified as is the direct precursor of teasterone. By feeding experiments with deuterium-labeled teasterone, it was shown to be converted to typhasterol. This epimerization would occur via oxidation to the 3-oxo-form. 3-Dehydrotesterone was shown to be reduced to typhasterol as major metabolite and to teasterone as minor one (Suzuki, 1995). The conversion of typhasterol to Cs and Bl was demonstrated by feeding with deuterium-labeled substrate. Typhasterol was predominantly converted to castasterone, while minor epimerization to teasterone was observed. Convertion of Cs to Bl was at first shown by feeding with deuterium-labeled castasterone (Suzuki et al., 1993), and later conclusively demonstrated by feeding with tritium-labeled Cs
 
3 - -
 INTRODUCTION
(Yokota et al., 1990). Thus, the biosynthesis of Bl via the early C6-oxidation pathway was described as shown in Figure 3.  
 Figure 2.Biosynthetic pathway from campesterol to 6-oxocampestanol  
 Figure 3.Biosynthesis of Bl via early C6-oxidation pathway.  Among natural BRs, attention was not drawn to the 6-deoxo BRs such as to 6-deoxocastasterone. They were considered to be dead-end in the pathway and not to be converted to active BR. However, reports showing the natural occurrence of 6-deoxo BRs in many plants (Abe et al., 1994; Yokota et al., 1994) have allowed their involvement in the biosynthetic pathway to be reevaluated. Feeding experiments with deuterium-labeled substrate revealed the conversion of 6-deoxoteasterone to 6-deoxotyphasterol. Similarly, 3-dehydro-6-deoxoteasterone was shown to be converted to 6-deoxotyphasterol. Thus, 6-deoxoteasterone is epimerized to 6-deoxotyphasterol via the 3-oxo form (3-dehydro-6-deoxoteasterone), similar to the convension of teasterone to typhasterol (Choi et al., 1997). Moreover, 6-deoxocastasterone was found to be converted to Cs and Bl (Choi et al., 1996). Therefore, the existence of an alternative Bl biosynthetic pathway via late C6-oxidation was demonstrated as shown in Figure 4. Both the early and the late C6-oxidation pathways operate in cell culture ofeus.rosC. The co-occurrence of BRs, belonging to the pathways in the same plant species indicates that these pathways could be ubiquitous in plants (Fujioka and Sakurai, 1997).  - 4 -